S-acylation of SARS-CoV-2 spike protein: Mechanistic dissection, in vitro reconstitution and role in viral infectivity

Abstract

S-acylation, also known as palmitoylation, is the most widely prevalent form of protein lipidation, whereby long-chain fatty acids get attached to cysteine residues facing the cytosol. In humans, 23 members of the zDHHC family of integral membrane enzymes catalyze this modification. S-acylation is critical for the life cycle of many enveloped viruses. The Spike protein of SARS-CoV-2, the causative agent of COVID-19, has the most cysteine-rich cytoplasmic tail among known human pathogens in the closely related family of β-coronaviruses; however, it is unclear which of the cytoplasmic cysteines are S-acylated, and what the impact of this modification is on viral infectivity. Here we identify specific cysteine clusters in the Spike protein of SARS-CoV-2 that are targets of S-acylation. Interestingly, when we investigated the effect of the cysteine clusters using pseudotyped virus, mutation of the same three clusters of cysteines severely compromised viral infectivity. We developed a library of expression constructs of human zDHHC enzymes and used them to identify zDHHC enzymes that can S-acylate SARS-CoV-2 Spike protein. Finally, we reconstituted S-acylation of SARS-CoV-2 Spike protein in vitro using purified zDHHC enzymes. We observe a striking heterogeneity in the S-acylation status of the different cysteines in our in cellulo experiments, which, remarkably, was recapitulated by the in vitro assay. Altogether, these results bolster our understanding of a poorly understood posttranslational modification integral to the SARS-CoV-2 Spike protein. This study opens up avenues for further mechanistic dissection and lays the groundwork toward developing future strategies that could aid in the identification of targeted small-molecule modulators. S-acylation, also known as palmitoylation, is the most widely prevalent form of protein lipidation, whereby long-chain fatty acids get attached to cysteine residues facing the cytosol. In humans, 23 members of the zDHHC family of integral membrane enzymes catalyze this modification. S-acylation is critical for the life cycle of many enveloped viruses. The Spike protein of SARS-CoV-2, the causative agent of COVID-19, has the most cysteine-rich cytoplasmic tail among known human pathogens in the closely related family of β-coronaviruses; however, it is unclear which of the cytoplasmic cysteines are S-acylated, and what the impact of this modification is on viral infectivity. Here we identify specific cysteine clusters in the Spike protein of SARS-CoV-2 that are targets of S-acylation. Interestingly, when we investigated the effect of the cysteine clusters using pseudotyped virus, mutation of the same three clusters of cysteines severely compromised viral infectivity. We developed a library of expression constructs of human zDHHC enzymes and used them to identify zDHHC enzymes that can S-acylate SARS-CoV-2 Spike protein. Finally, we reconstituted S-acylation of SARS-CoV-2 Spike protein in vitro using purified zDHHC enzymes. We observe a striking heterogeneity in the S-acylation status of the different cysteines in our in cellulo experiments, which, remarkably, was recapitulated by the in vitro assay. Altogether, these results bolster our understanding of a poorly understood posttranslational modification integral to the SARS-CoV-2 Spike protein. This study opens up avenues for further mechanistic dissection and lays the groundwork toward developing future strategies that could aid in the identification of targeted small-molecule modulators. The Coronavirus disease 2019 (COVID-19) pandemic, the most recent epidemic caused by an outbreak of zoonotic coronaviruses in the past two decades, was preceded closely by MERS in 2012 and SARS in 2003 (1Sharif-Yakan A. Kanj S.S. Emergence of MERS-CoV in the Middle East: Origins, transmission, treatment, and perspectives.PLoS Pathog. 2014; 10e1004457Crossref PubMed Scopus (66) Google Scholar, 2Cherry J.D. The chronology of the 2002-2003 SARS mini pandemic.Paediatr. Respir. Rev. 2004; 5: 262-269Crossref PubMed Scopus (102) Google Scholar). Since the onset of COVID-19, over 160 million cases have been recorded, resulting in more than 3.3 million deaths globally (3Dong E. Du H. Gardner L. An interactive web-based dashboard to track COVID-19 in real time.Lancet Infect. Dis. 2020; 20: 533-534Abstract Full Text Full Text PDF PubMed Scopus (6648) Google Scholar). These trends undoubtedly suggest that coronaviruses represent an ongoing threat to human health and economic stability for years to come (4Nicola M. Alsafi Z. Sohrabi C. Kerwan A. Al-Jabir A. Iosifidis C. Agha M. Agha R. The socio-economic implications of the coronavirus pandemic (COVID-19): A review.Int. J. Surg. 2020; 78: 185-193Crossref PubMed Scopus (4013) Google Scholar). As the causative agent of COVID-19, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has rightly received an unprecedented amount of attention from the scientific community and been the focus of intense investigation (5COVID research: A year of scientific milestones.Nature. 2021; https://doi.org/10.1038/d41586-020-00502-wCrossref Scopus (0) Google Scholar). However, several aspects about this virus remain poorly understood. These gaps in knowledge underscore the need to investigate the chemistry and biology of SARS-CoV-2 in a manner that could lead to a greater comprehension of the unknown aspects of the disease. As a global initiative to eradicate COVID-19 and fortify a future defense against similar pandemics, not only is it essential to focus research on the discovery of antivirals, but it is also important to obtain insights into the cell biology of SARS-CoV-2 and identify pathways with the highest potential for the discovery of novel therapeutics. SARS-CoV-2 belongs to the β-Coronavirus family of enveloped, positive-strand RNA viruses(6). Upon infection, the Spike (S) protein (Fig. 1A), resident in the viral membrane, directly mediates the critical process of membrane fusion, which is initiated upon binding to the angiotensin-converting enzyme 2 (ACE2) receptor located on the host cell plasma membrane (7Hoffmann M. Kleine-Weber H. Pohlmann S. A multibasic cleavage site in the spike protein of SARS-CoV-2 is essential for infection of human lung cells.Mol. Cell. 2020; 78: 779-784.e775Abstract Full Text Full Text PDF PubMed Scopus (1211) Google Scholar). Post infection, the viral genetic material encodes for several proteins along with the S protein, which traverse the ER-Golgi intermediate compartment (ERGIC) and Golgi where they get cleaved into the S1 and S2 proteins, that remain noncovalently attached to each other (8Nal B. Chan C. Kien F. Siu L. Tse J. Chu K. Kam J. Staropoli I. Crescenzo-Chaigne B. Escriou N. van der Werf S. Yuen K.Y. Altmeyer R. Differential maturation and of severe acute respiratory syndrome coronavirus proteins and PubMed Scopus Google Scholar). these as and get of the cell J. of coronavirus from proteins the from the of PubMed Scopus Google Scholar). SARS-CoV-2 S protein has been the most of research in the several most of the attention has been on the of S protein, resulting in a of and is known about the located on the of the has been that the of the S protein from closely related as and are targets of posttranslational S-acylation of S protein is for into and but not with PubMed Scopus Google Scholar, A. R. M. of the cysteine-rich of the spike is important for cell PubMed Scopus Google Scholar, A. of spike protein in coronavirus Full Text Full Text PDF PubMed Scopus Google the most prevalent form of modification of proteins M. van der PubMed Scopus Google Scholar). S-acylation, by the of long-chain fatty acids to cysteine residues to the of the membrane, is by 23 members of the zDHHC family of integral membrane enzymes in R. J.D. A. and of protein 2020; PubMed Scopus Google Scholar, protein stability and Rev. PubMed Scopus Google Scholar, H. Z. H. and Rev. PubMed Scopus Google Scholar). and proteins are targets of S-acylation, which is important for a of proteins have been as for zDHHC enzymes M. van der PubMed Scopus Google Scholar). Interestingly, S-acylation was in viral S proteins binding to virus A of modification of the viral Full Text PDF PubMed Scopus Google and is a of the S proteins in many enveloped RNA M. Differential S-acylation of enveloped PubMed Scopus Google Scholar). S-acylation has been to a for of the viral cycle a of virus in M. the identification of enzymes for of viral protein as potential 2020; PubMed Scopus Google mutation of S-acylation in the S protein of severely viral A the cytoplasmic of and SARS-CoV-2 that they are with the of an cysteine in the SARS-CoV-2 (Fig. the and of S-acylation in it is that is dissection of the S-acylation of viral protein that can a for the mechanistic understanding of this important we that the S protein of SARS-CoV-2 to as S is S-acylated, identify the of S-acylation, and in viral infectivity. we have developed a library of human zDHHC enzymes in expression in to identify members of the zDHHC family that are in S protein S-acylation. Finally, we in vitro the S-acylation of an S protein with purified zDHHC which into the of for further and lays the groundwork for the of novel for the identification of small-molecule M. L. N. N. C. of A for and PubMed Google Scholar, J. of protein S-acylation for the of 2020; PubMed Scopus Google Scholar). S-acylation cysteine residues that the cytosol. The cytoplasmic tail of SARS-CoV-2 S protein a of cysteines that to the (Fig. the S-acylation status of the cytoplasmic we an in cellulo chemistry assay. This was by of S protein in with a fatty by the and of a of protein in PubMed Scopus Google which S-acylation (Fig. A in which the cysteines in the S-acylation. The of a global of protein S-acylation, of S protein the of in this posttranslational modification (Fig. these that the S protein is by zDHHC enzymes more cysteine residues cytoplasmic of the cytoplasmic tail of the S proteins of SARS-CoV-2 with of and other closely related coronaviruses in to the cysteines in the SARS-CoV-2 S protein an cysteine (Fig. the of SARS-CoV-2 S protein to the most cysteine-rich in among the known human In SARS-CoV-2, these cysteines can into with each two cysteine residues and two with a cysteine (Fig. which of these are targets for S-acylation, we used the chemistry with S protein constructs that have each of the S-acylation by of the cysteine to (Fig. An a where the cysteine to SARS-CoV-2 S protein was to the of S protein and of the S protein in by S-acylation of the and with the of cysteines in S-acylation (Fig. of the a of with the S protein (Fig. further we the in which we from a in which the cysteines on the to on this each of the of cysteine was (Fig. in each of the constructs from only of the of cysteines was with our experiments, and the most of S-acylation, from the with the constructs (Fig. of was also We also for with a fatty that more closely A. and of fatty proteins using acids and Full Text Full Text PDF PubMed Scopus Google and is in the as a for results to with with and the site of S-acylation with a site (Fig. These that the S protein is and that the S-acylation status of the of cysteines the tail is the of the S protein on and S protein and in human and The to a of on in this The of infectivity. and by to was by about and of infectivity. The and a of (Fig. the mutation (Fig. investigate the of the on S protein and we of cell and virus from with the and the S protein expression A of was The a in of S1 in of (Fig. and however, of S2 to of (Fig. and The of of S2 severely of S1 the of S1 from the of The a similar of the and severe in the of S1 and S2 in The a of S protein expression and that was similar to that of the severely compromised (Fig. mutation of residues in the cytoplasmic tail of the S protein a of in was with in the of S1 S2 in for other was not to in expression The of S1 and S2 protein (Fig. We the effect of a of S protein and the of the S protein by with S protein expression with specific for the S protein and the The S protein a of with the the of the S protein and was similar to that of the (Fig. We also investigated the expression of and the and by using specific for the S1 and S2 of the S protein The results that these not S protein expression the cell (Fig. and viral pathogens not for enzymes that catalyze S-acylation. they on the S-acylation of the In humans, protein S-acylation is by 23 members of the zDHHC family of integral membrane enzymes that in as as the plasma zDHHC family of enzymes are in with for S-acylation. the zDHHC enzymes that S-acylate a need to The most prevalent by has been to a with each zDHHC M. H. of 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). The zDHHC members that the of S-acylation of a can as the zDHHC enzymes which on that the are of on that this has been with a library of by the M. H. of 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). this was of the of the as a However, in the of SARS-CoV-2, we it to a we the two of the of this with human the could more to human disease. 23 into the A. I. S. E. and expression of membrane proteins in for 2014; PubMed Scopus Google and for with a of of S protein with each human zDHHC enzymes that S protein (Fig. S protein expression over a when with different zDHHC members (Fig. is in the of cases where zDHHC enzymes for a specific have been by this S-acylation has been in to protein by protein van der The of protein on and Rev. PubMed Scopus Google Scholar). further our we the of the S protein with each zDHHC enzyme and (Fig. This the of the zDHHC members to S-acylate the S protein and further our from the of S protein with S protein S-acylation with the not (Fig. the of the not the S-acylation of S protein (Fig. Altogether, these and as S-acylation enzymes for the SARS-CoV-2 S protein. An essential the mechanistic dissection of S-acylation of protein is the of in vitro using purified proteins, that with can However, of in vitro of S-acylation by zDHHC enzymes in the S. L. and plasma membrane of by a in PubMed Scopus Google Scholar, R. A. and by an integral membrane PubMed Scopus Google most of them with of S. The cysteine-rich of the is and Full Text Full Text PDF PubMed Scopus Google Scholar). 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We human and human using the same in vitro S-acylation assay. we S-acylation of the S protein in with the and a similar of heterogeneity among the of S-acylation in the different (Fig. and However, the different zDHHC members a of among the and the highest with and the highest with these that and and the highest of S-acylation in with our in cellulo S-acylation of viral proteins has been known the discovery of this binding to virus A of modification of the viral Full Text PDF PubMed Scopus Google Scholar). SARS-CoV-2 S protein the most cysteine-rich tail with other related coronaviruses as and MERS-CoV (Fig. However, it has been unclear of these cysteines are and that impact viral infectivity. we that the S protein of SARS-CoV-2 is and identify the of S-acylation. 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This into our results when that that the viral spike protein for A is with a fatty most on cytoplasmic tail for the site to the which is with an fatty L. van der M. of but not of and is by and J. 2020; PubMed Scopus Google Scholar). suggest a for heterogeneity by that which fatty acids are of to the for three zDHHC and and cysteine clusters the highest of S-acylation. These not only a for an in vitro of viral protein S-acylation with purified zDHHC enzymes but also the for further of the mechanistic of S-acylation of SARS-CoV-2 S protein. could the of S-acylation of S protein in the life cycle of S-acylation is known to proteins to membrane as has been for of S protein is for into and but not with PubMed Scopus Google Scholar). it has been for other enveloped that is an important of the of membrane by E. H. A. C. J. to membrane in A virus and membrane PubMed Scopus Google to S protein in as as a of viral with the membrane membrane of PubMed Scopus Google Scholar, J. L. 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I. for viral and 2021; Google Scholar). we also that the form of S protein was compromised in to into and could to the to the and of this process more on the S-acylation and the SARS-CoV-2 life In this it is that in with other related SARS-CoV-2 has the most cysteine-rich cytoplasmic Finally, these also S-acylation as a against viral zDHHC enzymes with been as a for several other J. of protein S-acylation for the of 2020; PubMed Scopus Google the of a small-molecule of S-acylation has not been The recent of and with the in vitro are the toward that zDHHC into from and of was using specific was purified using and with enzymes of with and into the using L. of up to several PubMed Scopus Google Scholar). enzymes in and and using and and from into with and was to the developed of SARS-CoV-2 was used as a The of the which the S-acylation of was with and a of protein as the constructs on the using and was developed by the E. S.S. in of a protein modification in S. A. PubMed Scopus Google in with and with and was of in a purified in was with in and to a of in the was to the and for with by with 2 with two in and in of with was initiated by with a using by in in for The by for The was with and on a with for from each a was on the expression of the A of zDHHC (1Sharif-Yakan A. Kanj S.S. Emergence of MERS-CoV in the Middle East: Origins, transmission, treatment, and perspectives.PLoS Pathog. 2014; 10e1004457Crossref PubMed Scopus (66) Google Scholar, 2Cherry J.D. The chronology of the 2002-2003 SARS mini pandemic.Paediatr. Respir. Rev. 2004; 5: 262-269Crossref PubMed Scopus (102) Google Scholar, E. Du H. Gardner L. An interactive web-based dashboard to track COVID-19 in real time.Lancet Infect. Dis. 2020; 20: 533-534Abstract Full Text Full Text PDF PubMed Scopus (6648) Google Scholar, M. Alsafi Z. Sohrabi C. Kerwan A. Al-Jabir A. Iosifidis C. Agha M. Agha R. The socio-economic implications of the coronavirus pandemic (COVID-19): A review.Int. J. Surg. 2020; 78: 185-193Crossref PubMed Scopus (4013) Google Scholar, research: A year of scientific milestones.Nature. 2021; https://doi.org/10.1038/d41586-020-00502-wCrossref Scopus (0) Google Scholar, J. J. B. M. M. L. The and of SARS-CoV-2 2020; PubMed Scopus Google Scholar, M. Kleine-Weber H. Pohlmann S. A multibasic cleavage site in the spike protein of SARS-CoV-2 is essential for infection of human lung cells.Mol. Cell. 2020; 78: 779-784.e775Abstract Full Text Full Text PDF PubMed Scopus (1211) Google Scholar, B. Chan C. Kien F. Siu L. Tse J. Chu K. Kam J. Staropoli I. Crescenzo-Chaigne B. Escriou N. van der Werf S. Yuen K.Y. Altmeyer R. Differential maturation and of severe acute respiratory syndrome coronavirus proteins and PubMed Scopus Google Scholar, J. of coronavirus from proteins the from the of PubMed Scopus Google Scholar, of S protein is for into and but not with PubMed Scopus Google Scholar, A. R. M. of the cysteine-rich of the spike is important for cell PubMed Scopus Google Scholar, A. of spike protein in coronavirus Full Text Full Text PDF PubMed Scopus Google Scholar, M. van der PubMed Scopus Google Scholar, R. J.D. A. and of protein 2020; PubMed Scopus Google Scholar, protein stability and Rev. PubMed Scopus Google Scholar, H. Z. H. and Rev. PubMed Scopus Google Scholar, binding to virus A of modification of the viral Full Text PDF PubMed Scopus Google Scholar, M. Differential S-acylation of enveloped PubMed Scopus Google Scholar, M. the identification of enzymes for of viral protein as potential 2020; PubMed Scopus Google Scholar, M. L. N. N. C. of A for and PubMed Google Scholar, J. of protein S-acylation for the of 2020; PubMed Scopus Google Scholar, of protein in PubMed Scopus Google Scholar, A. and of fatty proteins using acids and Full Text Full Text PDF PubMed Scopus Google Scholar, M. 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